Structural basis for photoswitching in fluorescent proteins brought into focus

Apr 10, 2007
Fluorsence: On and Off
Graphic shows models of the on and off structural alignments of a photoswitchable fluorescent protein. Credit: Courtesy S. James Remington

University of Oregon scientists have identified molecular features that determine the light-emitting ability green fluorescent proteins, and by strategically inserting a single oxygen atom they were able to keep the lights turned off for up to 65 hours.

The findings, published online this week by the Proceedings of the National Academy of Sciences, likely are applicable to most photoswitchable fluorescent proteins, said S. James Remington, professor of physics and member of the UO Institute of Molecular Biology.

"This new model makes specific predictions and improves the qualities of the protein as a photo-switchable label," Remington said. "It gives us the first picture of how these molecules can be switched on and off. That allows us to design new variants to make the proteins more useful."

For more than a decade, fluorescent proteins – first isolated in jellyfish and since found in a variety of colors from coral reef organisms – revolutionized molecular biology, allowing scientists to use them as markers for genetic expression, to locate molecules and observe activity within cells.

The recent discovery of photoswitchable fluorescent proteins – which can be manipulated with a laser – has been a significant development for cellular research.

"Photoswitchable fluorescent proteins have tremendous advantages over passive proteins," Remington said. "You can label all molecules but using a laser under a microscope, you can activate only a small group of them. That lets you follow the motion of subsets of molecules. We wanted to understand the process, so that we can permanently switch them off and on or vary the time delay."

However, he said, the mechanism of photoswitching was unknown, and in many cases the proteins returned to their stable state randomly and spontaneously.

Using a combination of rational mutagenesis and directed evolution, UO doctoral student J. Nathan Henderson determined high-resolution crystal structures of both the on and off states of a fluorescent protein isolated from a sea anemone.

In the stable or fluorescent state of the molecule, two side chains of atoms align in a coplanar fashion, flat and in orderly fashion. When hit with bright laser light, the researchers observed that the protein rapidly went dark as the rings rotated about 180 degrees and flip by some 45 degrees, coming to rest in a non-coplanar and unstable alignment. The two structures gave the researchers a chance to observe changes in the interactions between neighboring groups.

Remington said that in the dark state, the molecule absorbs ultraviolet light and doesn't emit any light at all. However, when the chromophore (a group of atoms and electrons forming part of an molecule) absorbs ultraviolet light, it occasionally ionizes and become negatively charged. This causes the rings to flip back into the fluorescent state.

Having control of light emission would allow for more precise studies within cells, he said.

Henderson studied the structures, noticing that in the dark state there was an unfavorable interaction where carbon and oxygen atoms were adjacent to each other. "Nathan looked at this and wondered what would happen if an oxygen atom was inserted at a precise place," Remington said. "That would make for a favorable interaction that stabilized the dark state. Based on the structure, Henderson made a single mutation that delays the switch-on time from five minutes to 65 hours.

Eventually, he added, the ability to control the on-off states could lead to improvements in optical memory, such as single molecule information storage, in addition to enhancing microscopic work and molecular labeling.

Source: University of Oregon

Explore further: How the hummingbird achieves its aerobatic feats

add to favorites email to friend print save as pdf

Related Stories

Optical control of motor functions

Nov 10, 2014

MIT researchers have demonstrated a highly flexible neural probe made entirely of polymers that can both optically stimulate and record neural activity in a mouse spinal cord—a step toward developing prosthetic ...

Molecular beacons shine light on how cells 'crawl'

Oct 24, 2014

Adherent cells, the kind that form the architecture of all multi-cellular organisms, are mechanically engineered with precise forces that allow them to move around and stick to things. Proteins called integrin ...

Extremely high-resolution magnetic resonance imaging

Oct 21, 2014

For the first time, researchers have succeeded to detect a single hydrogen atom using magnetic resonance imaging, which signifies a huge increase in the technology's spatial resolution. In the future, single-atom ...

Recommended for you

How the hummingbird achieves its aerobatic feats

10 hours ago

(Phys.org) —The sight of a tiny hummingbird hovering in front of a flower and then darting to another with lightning speed amazes and delights. But it also leaves watchers with a persistent question: How ...

New terahertz device could strengthen security

Nov 21, 2014

We are all familiar with the hassles that accompany air travel. We shuffle through long lines, remove our shoes, and carry liquids in regulation-sized tubes. And even after all the effort, we still wonder if these procedures ...

CERN makes public first data of LHC experiments

Nov 21, 2014

CERN today launched its Open Data Portal where data from real collision events, produced by experiments at the Large Hadron Collider (LHC) will for the first time be made openly available to all. It is expected ...

User comments : 0

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.